Faculty of Agricultural Sciences, Department of Phytopathology, Institute of Phytomedicine, University of Hohenheim, Stuttgart, Germany.
PLoS One. 2021 Sep 10;16(9):e0257225. doi: 10.1371/journal.pone.0257225. eCollection 2021.
Diaporthe species are fungal plant pathogens of many important crops. Seed decay is one of the most important diseases on soybean. It is caused by various species of the genus Diaporthe and responsible for significant economic damage. In central Europe the four species D. longicolla, D. caulivora, D. eres, and D. novem are considered the principal species of Diaporthe on soybean. Fast and accurate detection of these pathogens is of utmost importance. In this study four species-specific TaqMan primer-probe sets that can be combined into a quadruplex assay were designed based on TEF sequences. The specificity and efficiency of the primer-probe sets were tested using PCR products and genomic DNA from pure cultures of the four Diaporthe species and other soybean fungal pathogens. Our results indicate that the primer-probe sets DPCL, DPCC, DPCE, and DPCN allow discrimination of D. longicolla, D. caulivora, D. eres, and D. novem, respectively, and can be used to detect and quantify these four Diaporthe species in parallel using quadruplex real-time PCR. In addition, the quadruplex real-time PCR assay was evaluated on different plant materials including healthy and infected soybean seeds or seed lots, soybean stems, and soybean leaves. This assay is a rapid and effective method to detect and quantify Diaporthe species from samples relevant for disease control.
层出孔菌属真菌是许多重要作物的植物病原菌。种子腐烂是大豆最重要的病害之一。它是由层出孔属的各种物种引起的，对经济造成了重大损失。在中欧，D. longicolla、D. caulivora、D. eres 和 D. novem 这四个物种被认为是大豆上的主要层出孔菌物种。快速准确地检测这些病原体至关重要。在这项研究中，基于 TEF 序列设计了四个种特异性的 TaqMan 引物-探针组合，可以组合成四重 PCR 检测。使用来自四个层出孔菌物种和其他大豆真菌病原体的纯培养物的 PCR 产物和基因组 DNA 测试了引物-探针的特异性和效率。我们的结果表明，引物-探针组合 DPCL、DPCC、DPCE 和 DPCN 分别允许区分 D. longicolla、D. caulivora、D. eres 和 D. novem，并可用于使用四重实时 PCR 平行检测和定量这些四个 Diaporthe 物种。此外，还在不同的植物材料（包括健康和感染的大豆种子或种子批、大豆茎和大豆叶片）上评估了四重实时 PCR 检测。该检测方法是一种快速有效的方法，可从与疾病控制相关的样品中检测和定量层出孔菌物种。
